Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe.

Abstract

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca ~+ and Mg ~+ ions bound to membranes. Acini, preloaded by incubation with CTC (100/xM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 rain of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg 2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secret~ agogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca~+-complexed CTC.KEY WORDS exocrine pancreas stimulussecretion coupling calcium fluorescent probe chlorotetracyclinePancreatic acinar cells are thought to use calcium as a second messenger because the divalent cation ionophore, A23187, is able to increase enzyme release from these cells in the presence of extracellular calcium (10,16,32,39). However, the manner in which physiological secretagogues initiate a rise in cytosol calcium is not known.

Thyrotropin-releasing Hormone Stimulation of Prolactin Release from Clonal Rat Pituitary Cells

“…Although other factors could have affected ieared to mobilize Ca2+ only from a pool (or CTC fluorescence, the very close parallels betweeni was monitored completely by incubating the effects of TRH on 45Ca2+ efflux and CTC fluoreswith 100 ,M CTC for 10 min. This con-cence suggest that the decrease in fluorescence caused s based on1 the finding that TRH caused a by TRH is best explained by displacement of memolute decrease in CTC fluorescence irre-brane-bound Ca2+, as has been concluded for other the duration of incubation with CTC be-secretagogues (28)(29)(30)(31)(32)(33)(34)(35). nd 120 mim, even though the total fluoresIt has been suggested that stimulation of release of isity increased with longer inctubations (data adenohypophyseal hormones may be dependent on Hence, it seems likely that TRH mobilized extracellular Ca2+ (36,37).…”

Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg2+

“…We chose 50 AM CTC as a standard concentration for all subsequent studies. In previous studies with other organelles and cells, the CTC concentration used to measure membrane-associated Mg" and Ca>2 has varied from 10 ,M (4,24) up to 100 ,uM (6,23).…”

“…The fluorescence emission of CTC2 has been used to measure the level of divalent cations (specifically Mg2" and Ca2") associated with envelope membranes in a wide range of animal cells and subcellular particles such as erythrocytes (23), pancreatic acinar (6) and islet (24) cells, brain synaptosomes (3), liver mitochondria (4, 16), and sarcoplasmic reticulum vesicles from skeletal muscle (18). The quantum yield of the complex increases in apolar, in comparison to aqueous environments (16).…”

Biparametric fluorometry of chlorotetracycline and ethidium bromide in DNA structure.

“…We examined whether the two fluorescent probes competed with each other for the DNA strand structure. CTC chelates Mg2+ as well as Ca2+ and both chelate compounds caused a spectral shift of the fluorescent emission of CTC (2,3,12). From the spectra of EB and CTC, we found that the emissions could be recorded separately by measuring F590 excited at 510 nm for EB, F520 excited at 400 nm for CTC-Mg and F420 excited at 350 nm for CTC alone, although there was some spectral overlapping of these spectra.…”